Method and composition for detecting bacterial contamination in food products

ABSTRACT

This invention relates to a method for detecting the existence or measuring the concentration of total viable bacteria in a test sample from a food product. A medium is provided which contains three or more different enzyme substrates each having a nutrient moiety and a detectable moiety linked together. When a substrate is hydrolysed by a bacterial enzyme to create a separate detectable moiety, it causes or produces a detectable signal. These substrates produce detectable signals when any one of a phosphatase enzyme, a glycosidase enzyme or a peptidase enzyme is present in the medium.

FIELD OF THE INVENTION

[0001] This invention relates to methods and compositions for detectingthe existence or measuring the concentration of bacterial contaminationin food products.

BACKGROUND OF THE INVENTION

[0002] Ground beef and chicken are susceptible to rapid spoilage bypsychotropic bacteria which thrive at refrigeration temperatures. As aresult, these products have very short shelf-lives which are directlyrelated to the initial concentration of contaminating bacteria.

[0003] Current methods for measuring the concentrations of bacterialcontamination in ground beef and chicken include the standard platecount (Difco Laboratories) as well as the Petri Film system (3M) (seegenerally, Compendium of Methods for the Microbiological Examination ofFoods, Third Edition, Edited by Carl Vanderzant and Don F.Splittstoesser, Compiled by the APHA Technical Committee onMicrobiological Methods for Foods). These methods require around 48hours of incubation in a 35° C. incubator before the results can beread. Both methods utilize a solid nutrient base to support the growthof individual cells into bacterial colonies. Many food-borne bacteriaare incapable of growing into colonies on these surfaces when incubatedat 35° C.; thus, the concentrations of total viable bacteria measured bythe above methods may be underestimated.

[0004] In addition, the long incubation periods of these methods cancause these food products to remain in storage for several days untilthe concentrations of contaminating bacteria are known. If these testscould be completed in a shorter period of time it would allow companiesto release their products sooner so as to lower costs, increase sales,and provide better product to the consumer.

[0005] There have been attempts to measure the bacterial concentrationin food by measuring specific metabolic by-products of individualmicroorganisms. These methods include: electrical impedance assays, ATPassays, antibody-based assays, and carbon-14 labelled substrate assays.Indicators of microbial growth have also been used to monitor the growthof target microbes which change color only after growth of the targetmicrobe is detected. These indicators normally react chemically with ametabolic by-product produced by the is target microbes resulting in acolor change in the medium. Examples of chemicals which change color inthe presence of pH changes associated with growth include phenol red,bromocresol blue, and neutral red. For example, Golber, U.S. Pat. No.3,206,317, uses phenol red, a chemical which changes color in thepresence of acidic waste products produced by the target microbe. Bergeret al., U.S. Pat. No. 3,496,066, describes the use of compounds whichbacteria convert to dyestuffs, e.g., tropinones and dioxanes, Bochner,U.S. Pat. No. 4,129,483 describes using a non-biodegradable substance(tetrazolium) which is chemically reduced to produce a color change. Inall of these examples, the indicator is a compound which does not serveas a source of a required nutrient.

[0006] Edberg (U.S. Pat. No. 4,925,789), incorporated by referenceherein, describes a selective growth medium for a microbe containing anutrient indicator which can only be metabolized by a target microbe.When metabolized by a target microbe, the nutrient indicator releases amoiety which imparts a detectable change to the medium.

SUMMARY OF THE INVENTION

[0007] The present invention relates to a bacterial growth medium andmethods for detecting the existence or measuring the concentration ofbacteria in a test sample. The claimed medium and methods measure viablebacteria as a function of the activities of several classes of bacterialenzymes, including, but not limited to, phosphatases, glycosidases (suchas glucosidases), and aminopeptidases. The presence of at least one ofthese groups of enzymes in any given bacterial species will be detectedby the appearance of a detectable signal such as a fluorescent signal.Therefore, this invention is useful in detecting the existence ormeasuring the concentration of total viable bacteria or at least amultitude of viable bacteria in a test sample in a single assay. Inspecific examples, cocktails of enzyme substrates are made to measurethe concentration of bacterial contamination in food products, such asground beef and chicken.

[0008] Thus, in one aspect, the invention features a bacterial growthmedium containing three or more different enzyme substrates eachhydrolysed by a different bacterial enzyme to cause or produce adetectable signal.

[0009] In a preferred embodiment, the three or more different enzymesubstrates each has both a nutrient moiety and a detectable moietylinked together by a covalent bond. Each of these enzyme substrates ishydrolysed by a different bacterial enzyme to produce a separatedetectable moiety which causes or produces a detectable signal in themedium. In a further preferred embodiment, the detectable signals causedor produced are of identical type.

[0010] By “medium” is meant a solid, powder or liquid mixture whichcontains all or substantially all of the nutrients necessary to supportbacterial growth. Amino acids, minerals, vitamins and other elementsknown to those skilled in the art to be necessary for bacterial growthare provided in the medium, including, but not limited to, thosedisclosed in U.S. application Ser. Nos. 08/334,788 and 08/335,149, bothfiled on Nov. 4, 1994, incorporated by reference herein. In a preferredembodiment, the medium is liquid.

[0011] For example, the following components are provided in the mediumin approximately the amounts indicated. Those in the art will understandthat not every component is required. Components may also be substitutedwith other components of similar properties. The amounts of componentsmay also be varied.

[0012] Amino acids may be provided from a variety of sources. These canbe provided from natural sources (e.g., extracts of organisms), asmixtures, or in purified form. The natural mixtures may contain varyingamounts of such amino acids and vitamins. Not all amino acids must beprovided, and the relative amount of each can vary. For generalguidance, specific amounts of such amino acids and vitamins areindicated below. These amounts are for guidance only and are notlimiting in this invention. Those in the art will recognize that manydifferent combinations of amino acids and vitamins can be used in themedium of this invention. The lists provided below exemplify just onesuch example. Normally, only amino acids which cannot be synthesizedendogenously by the microorganisms to be detected must be provided.However, other amino acids may be provided without departing from themedium of the invention.

[0013] The medium preferably includes at least the following amino acidsin approximately the following amounts (per liter of medium): Alanine(0.015 to 0.60 grams), Arginine (0.080 to 3.2 grams), Aspartic Acid(0.018 to 0.72 grams), Cystine (0.09 to 3.6 grams), Glutamic Acid (0.030to 1.20 grams), Glycine (0.050 to 2.00 grams), Histidine (0.025 to 1.00grams), Isoleucine (0.035 to 1.40 grams), Leucine (0.040 to 1.60 grams),Lysine (0.050 to 2.00 grams), Methionine (0.01 to 0.50 grams),Phenylalanine (0.01 to 0.90 grams), Proline (0.02 to 2.80 grams), Serine(0.01 to 0.40 grams), Threonine (0.01 to 1.10 grams), Tryptophan (0.002to 0.26 grams), Tyrosine (0.01 to 1.20 grams), and Valine (0.02 to 1.10grams).

[0014] Salts may be provided as a source of ions upon dissociation. Suchsalts may include (per liter of medium): potassium chloride (e.g., about0.5 to 1.5 grams); copper sulfate (e.g., about 40 to 50 μg); ammoniumacetate or ammonium sulfate (e.g., about 4.0 to 6.0 grams); potassiumiodide (e.g., about 50.0 to 150.0 μg); ferric chloride (e.g., about150.0 to 250.0 μg); manganese sulfate (e.g., about 300.0 to 500.0 μg);sodium molybdate (e.g., about 150.0 to 250.0 μg); zinc sulfate (e.g.about 300.0 to 500.0 μg); and sodium chloride (e.g. about 0.05 to 0.15g).

[0015] Other inorganic moieties may be included to aid microbial growth.These include the following (to the extent not already provided in theabove sources of various chemical entities and described in amounts perliter): Phosphorus (about 0.5 mg), Potassium (about 0.4 mg), Sodium(about 30 to 60 mg), and trace amounts of Calcium, Magnesium, Aluminum,Barium, Chloride, Cobalt, Copper, Iron, Lead, Manganese, Suffate,Sulfur, Tin and Zinc.

[0016] Vitamins required for growth and reproduction of themicroorganism sought to be detected may also be provided. These can beprovided in a pure form or as part of a more complex medium. Suchvitamins may be present in approximately the following amounts (perliter of medium): Biotin (about 0.15 to 60 μg), Pantothenic Acid (about15.0 to 65.0 μg), Pyridoxine (about 2.0 to 9.0 μg), Riboflavin (about10.0 to 50.0 μg), Folic acid (about 5.00 to 50.00 μg), Thiamine (about10.0 to 50.0 μg), Vitamin B12 (about 0.20 to 0.50 μg), and Niacin (about15.0 to 55.0 μg).

[0017] By “bacterial enzyme” is meant an enzyme whose enzymatic activitysuch as the ability to hydrolyse a substrate or a plurality ofsubstrates is characteristic of a bacterium or a plurality of bacteria.In this invention, the enzymatic activities of a bacterial enzyme orbacterial enzymes are used to detect or measure the concentration ofbacteria in a test sample. The bacterial enzymes include all those knownto one skilled in the art, including, but not limited to, those listedin Enzymes, 3rd edition, edited by Malcolm Dixson, Edwin C. Webb, C. J.R. Thorne, and K. F. Tipton, 1979, Academic Press, U.S.A. In a preferredembodiment, the bacterial enzyme is selected from the group consistingof alkaline phosphatase, acid phosphatase, esterase, lipase,N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase,Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase,β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase,β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase,sulfatase, β-D-xylosidase, peptidase (preferably an aminopeptidase, morepreferably an (L or D amino acid)-aminopeptidase), trypsin,chymotrypsin, and phosphohydrolase.

[0018] By “substrate” is meant a molecule or substance on which abacterial enzyme acts. The enzymatic reaction usually involveshydrolysing one or more covalent bonds, forming one or more covalentbonds, or both. A covalent bond in the substrate between the nutrientmoiety and the detectable moiety is hydrolysed by a bacterial enzyme toproduce a separate detectable moiety. The substrates include all thoseknown to one skilled in the art, including, but not limited to, those inthe product listing of AerChem, Inc. with detectable moieties attachedthereto (see Table I).

[0019] By “nutrient moiety” is meant a molecule or substance which is anutrient or metabolic source for a bacterium, including, but not limitedto, vitamins, minerals (e.g., phosphorus in the form of phosphate),trace elements, amino acids (e.g., L-alanine), carbon (e.g., glucose),or nitrogen.

[0020] By “detectable signal” is meant a characteristic change in amedium or sample that is observable or measurable by physical, chemical,or biological means known to those skilled in the art. Such a detectablesignal may be a change in emission or absorbance of visible or invisiblelight or radio waves at a certain wavelength, electrical conductivity,hybridization, enzymatic reaction, emission of gas, or odor. Adetectable signal may also be a change in physical state such as betweensolid, liquid and gas. In preferred embodiments, detectable signalsinclude a change in color or fluorescent emission of the medium.

[0021] By “identical type of detectable signal” is meant that theseparate detectable moieties hydrolysed from different enzyme substratescause or produce detectable signals that are measurable by the same orsubstantially the same physical, chemical or biological parameter,including, but not limited to, color, fluorescent emission, odor,enzymatic reaction, hybridization, or electric conductivity (althoughthe intensity or quantity of signals caused or produced by differentseparate detectable moieties may be different). For example, yellowcolors of different intensity would be considered of the identical type.Color change and fluorescence would not be considered to be identicaltype of detectable signal.

[0022] By “detectable moiety” is meant a molecule or substance which canbe covalently linked to a nutrient moiety or exists as a separate entityby itself. The detectable moiety does not cause or produce a detectablesignal when it is covalently bonded to a nutrient moiety. However, whenan enzyme from a bacterium hydrolyses the substrate, a detectable moietyis released and causes or produces a detectable signal. In preferredembodiments, the detectable moieties are chromogens which produce acolor change observable in the visible wavelength range or fluoresceswhen properly excited by an external energy source. Examples ofdetectable moieties include, but are not limited to, orthonitrophenyl,phenolphthalein, and 4-methylumbelliferone moieties.

[0023] The invention also features a method of using the medium todetect the existence or measure the concentration of bacterialcontamination in a test sample. The medium is inoculated with the testsample and incubated under a condition suitable for bacterial growth fora certain time period (preferably no more than 24 hours, more preferablyno more than 15 hrs, even more preferably no more than 10 hours). Thenthe detectable signal is measured as an indication of the concentrationof bacteria in the test sample. Using this method, a detectable signalis produced when at least one of the three or more different bacterialenzymes is or are present in the bacteria which are incubating in themedium.

[0024] By “test sample” is meant a piece, fraction, aliquot, droplet,portion, fragment, volume, or tidbit taken from a food product such asground beef or chicken, a human or animal test subject, a soil, water,air or other environmental source, or any other source whose bacterialconcentration is to be measured. A test sample may be taken from asource using techniques known to one skilled in the art, including, butnot limited to, those described or referred to in Compendium of Methodsfor the Microbiological Examination of Foods, Third Edition, Edited byCarl Vanderzant and Don F. Splittstoesser, Compiled by the APHATechnical Committee on Microbiological Methods for Foods, incorporatedby reference herein.

[0025] By “bacteria” is meant one or more viable bacteria existing orco-existing collectively in a test sample. The term may refer to asingle bacterium (e.g., Aeromonas hydrophilia, Aeromonas caviae,Aeromonas sobria, Streptococcus uberis, Enterococcus faecium,Enterococcus faecalis, Bacillus aphaericus, Pseudomonas fluorescens,Pseudomonas putida, Serratia liquefaciens, Lactococcus lactis,Xanthomonas maltophilia, Staphylococcus simulans, Staphylococcushominis, Streptococcus constellatus, Streptococcus anginosus,Escherichia coli, Staphylococcus aureus, Mycobacterium fortuitum, andKlebsiella pneumonia), a genus of bacteria (e.g., streptococci,pseudomonas and enterococci), a number of related species of bacteria(e.g. coliforms), an even larger group of bacteria having a commoncharacteristic (e.g., all gram-negative bacteria), a group of bacteriacommonly found in a food product, an animal or human subject, or anenvironmental source, or a combination of two or more bacteria listedabove. The bacteria include those described or referred to in Bergey'sManual of Systematic Bacteriology, 1989, Williams and Wilkins, U.S.A.,incorporated by reference herein.

[0026] In preferred embodiments, one of the substrates is hydrolysed bythe enzyme alkaline phosphatase; another substrate is hydrolysed by theenzyme glycosidase, including, but not limited to, β-D-glucosidase; anda third substrate is hydrolysed by a peptidase (preferably anaminopeptidase, more preferably an (L or D amino acid)-aminopeptidase),including, but not limited to, L-alanine aminopeptidase; the detectablemoiety is a fluorescent moiety such that when the detectable moiety ishydrolysed from a substrate, it causes or produces a fluorescent signal;the medium contains at least the following three substrates:4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucoside andL-alanine-7-amido-4-methyl coumarin; and the medium is inoculated with atest sample from a food product, including, but not limited to, groundbeef, chicken, milk, dairy products, and drinking water.

[0027] In another aspect, the invention features a bacterial growthmedium containing two or more different enzyme substrates eachhydrolysed by a different bacterial enzyme to cause or produce anidentical type of detectable signal.

[0028] In a preferred embodiment, the two or more different substrateseach has both a nutrient moiety and a detectable moiety linked togetherby a covalent bond. Each of these substrates is hydrolysed by adifferent bacterial enzyme to produce a separate detectable moiety whichcauses or produces an identical type of detectable signal.

[0029] The invention also features a method of using the medium todetect the existence or measure the concentration of bacteria in a testsample. The medium is inoculated with the test sample and incubatedunder a condition suitable for bacterial growth for a certain timeperiod (preferably no more than 24 hours, more preferably no more than15 hrs, even more preferably no more than 10 hours). Then the detectablesignal is measured as an indication of the concentration of bacterialcontamination in the test sample. Using this method, a detectable signalis produced when at least one of the two or more different bacterialenzymes is present in the incubation medium.

[0030] In preferred embodiments, the substrates are hydrolysed by anenzyme selected from the group consisting of alkaline phosphatase,glycosidase (which includes, but is not limited to, β-D-glucosidase),and peptidase (preferably an aminopeptidase, more preferably an (L or Damino acid)-aminopeptidase, including, but not limited to, L-alanineaminopeptidase); and the detectable moiety and the medium are analogousto those noted above.

[0031] In other embodiments, the invention uses the apparatus describedby Naqui et al. in U.S. patent application Ser. No. 08/201,110,incorporated by reference herein, to quantify the concentration ofbacterial contamination. An example of such an apparatus is sold byIdexx Laboratories Inc. under the name of Quanti Tray™. The quantifyingstep involves providing a test sample in a liquid form. The sample isplaced or dispensed into the sample holding bag described by Naqui etal., and mixed with a medium to allow and promote growth of targetbacteria within individual compartments. The mixture is incubated andthe quantity and quality of the color or fluorescence change in eachcompartment is detected. The quantity and quality of positivecompartment (i.e., a compartment having a detectable color orfluorescence change) is compared to a most probable number table whichrelates that value to the bacterial concentration of the test sample.

[0032] This invention has many advantages over the methods currentlyused to measure bacterial contamination. One advantage is its relativelyshort time to results. Certain psychotropic bacteria grow very slowlyand can take from 48 to 72 hours before their colonies become largeenough to count on an agar plate. However, countable colonies need notbe present for the results of Applicant's test to be read. Thefluorescent color produced by these bacteria in the invention appearsmuch faster than their corresponding colonies which results in a muchshorter detection time. Applicant's test can reduce the incubationperiod to 24 hours or less.

[0033] Another advantage of the invention has over standard methods isthe absence of interference by bacterial overgrowth. This is aparticular problem when Bacillus species are present because they tendto grow over other bacterial colonies in such a way that the plate isunreadable. The Bacillus species are common in food, particularly thosethat have been heat treated, such as pasteurized milk. This problem isavoided in the invention because it does not depend on countingindividual bacterial colonies.

[0034] This invention can be used in microbiology laboratories involvedin end product testing and/or quality control of food products; the meatand poultry industries, the dairy industry, and the water industry. Theinvention may be used to measure the concentration of total viablebacteria in drinking water.

[0035] This invention also relates to a growth medium and methods fordetecting or measuring the concentration of yeasts, fungi, or othereukaryotic microorganisms in a test sample using a formulated medium andsteps like those described above.

[0036] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments, and fromthe claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] In the following description, reference will be made to variousmethodologies known to those of skill in the chemical, biological andmicrobiological arts. Publications and other materials setting forthsuch known methodologies to which reference is made are incorporatedherein by reference in their entireties as though set forth in full. Thecompositions, methods, and products of this invention are applicable tobiological and environmental specimens, and are useful in the chemical,biological and microbiological arts for the detection of bacterialcontamination.

[0038] Detecting Bacteria by Measuring Bacterial Enzyme Activities

[0039] Bacteria derive their nutrients from an array of sources. Theability to metabolize certain sources may be characteristic of aparticular bacterium or group of bacteria. Families, groups or speciesof bacteria may share enzyme specificity for certain nutrients which arelacking in other bacteria. By taking advantage of the metaboliccharacteristics of bacteria, it is possible to test for the presence ofthese enzyme systems, and thus, the bacteria which display these enzymesystems themselves. See Edberg, supra. Many enzymes have been identifiedwhich are specific to particular groups of bacteria and others likelywill be identified in the future (see generally, Bergey's Manual ofSystematic Bacteriology, 1989, Williams and Wilkins, U.S.A.).

[0040] For example, most gram negative bacteria, as a group, haveL-alanine aminopeptidase enzyme activity. Substrates such asL-alanine-β-orthonitrophenyl, β-naphthalamide-β-L-alanine,α-naphthol-β-L-alanine, 4-methylumbelliferyl-β-L-alanine, andL-alanine-7-amido-4-methyl coumarin may be used in the medium to testfor the presence of gram negative bacteria. The enzyme β-D-glucosidaseis found in the Enterococcus group of bacteria. The enzyme may catalyzethe hydrolysis of 10 appropriate substrates containing chromogenic orfluorogenic moieties linked to a β-glucoside. This property may be usedto indicate the presence or absence of enterococci in a sample.Substrates such as 4-methylumbelliferyl-β-D-glucopyranoside may be usedto indicate the presence of enterococci. Staphylococcus aureus iscapable of hydrolysing orthonitrophenyl phosphate. Thus, if the growthmedium contains this substrate as a source of phosphate, Staphylococcusaureus will grow and a color change will be produced by the release ofthe orthonitrophenyl moiety. Mycobacterium fortuitum requires SO₄ as itssource of sulfur, and this species can hydrolysephenolphthalein-sulfate. Thus, in a selective medium whose only sulfursource is phenolphthalein-sulfate, this species will grow and produce acharacteristic color change by release of the colored moiety.Furthermore, the enzyme β-D-glucuronidase is present in E. coli.Substrates such as orthonitrophenyl-β-D-glucuronide,β-naphthalamide-β-D-glucuronide, α-naphthol-β-D-glucuronide ormethylumbelliferyl-β-D-glucuronide may be used in a medium for thedetection of E. coli.

[0041] Substrates and Detectable Moieties

[0042] Substrates including a chromogenic moiety have been demonstratedto display a characteristic color change in samples containing targetbacteria having a bacterial enzyme capable of hydrolysing thesubstrates. For example, in the presence of β-D-glucuronidase,orthonitrophenyl-β-D-glucuronide produces a color change to yellow,4-methylumbelliferyl-β-D-glucuronide produces fluorescence afterexcitation at 366 nm, and bromochloro-indole-β-D-glucuronide produces acolor change to blue when E. coli is present. In the presence ofβ-D-galactosidase, orthonitrophenyl-β-D-galactopyranoside produces acolor change to yellow and 4-methylumbelliferyl-β-D-galactopyranosideproduces fluorescence after excitation at 366 nm when E. coli ispresent.

[0043] Two substrates producing different types of detectable signalshave been used for detecting the presence of E. coli among totalcoliform bacteria. 4-methylumbelliferyl-β-D-glucuronide may be usedtogether with orthonitrophenyl-β-D-galactopyranoside. If any E. coli ispresent, the sample solution both changes color to yellow and emitsfluorescence after excitation at 366 nm.

[0044] Table I is a list of substrates from AerChem, Inc. that may beused to detect bacterial enzyme activities.

[0045] A detectable moiety may be attached to a nutrient moiety bymethods known to those skilled in the art. The methods generally featurecoupling or conjugating a nutrient moiety to a detectable moiety, suchas a chromogenic moiety. Examples of such methods are described byEdberg in U.S. Pat. No. 4,925,789, incorporated by reference herein.

[0046] The following non-limiting example features a liquid basedbacterial growth medium used to quantify the total number of viablebacteria present in ground beef and chicken. This medium comprises4-methylumbelliferyl phosphate (MUP), 4-methylumbelliferyl-β-D-glucoside(MUD), and L-alanine-7-amido-4-methyl coumarin (ala-AMC). An example ofthe composition is described in Table II. The composition of definedmedia is described in Table III. MUP, MUD, ala-AMC, and potassiumnitrate were purchased from Sigma. Bacto Proteose Peptone No. 3 waspurchased from DIFCO.

[0047] The substrate 4-methylumbelliferyl-β-D-glucoside is used todetect the presence of the enzyme β-D-glucosidase which is present inStreptococci, Enterococci, and other related bacteria commonly found infresh meat.

[0048] The substrate L-alanine-7-amido-4-methylcoumarin is used todetect the presence of the enzyme L-alanine aminopeptidase which isfound in most pseudomonas species and other gram negative bacteria.Applicant discovered that this substrate is particularly sensitive tothe presence of psychotropic bacteria which cause spoilage in meat.Other substrates can be used in place ofL-alanine-7-amido-4-methylcoumarin to detect other types ofaminopeptidases in this group of bacteria without sacrificingsensitivity.

[0049] The substrate 4-methylumbelliferyl phosphate is used to detectthe presence of phosphatases such as alkaline phosphatase and acidphosphatase which are found in most bacterial species. This enzymesubstrate supports the detection of bacteria which lack or havediminished L-alanine aminopeptidase and β-D-glucosidase activities.

[0050] Because phosphatase, β-D-glucosidase, and L-alanineaminopeptidase are present in the vast majority of bacteria whichcontaminate ground beef and chicken, only one of these enzymes needs tobe functional in the food-borne bacteria for viability to be detected.This test, therefore, has built-in redundant screens which support ahighly accurate measure of total viable bacteria in ground beef andchicken.

[0051] The presence of bacteria is indicated by the appearance of a bluefluorescent color in the medium after it is exposed to an externalultra-violet lamp (366 nm wavelength). This test yields result after nomore than 24 hours of incubation at 35° C.

[0052] The substrates MUP, MUD, or ala-AMC are hydrolysed byphosphatase, β-D-glucosidase, or L-alanine aminopeptidase to produceboth nutrient and fluorescent moieties. The nutrient moieties (i.e.,phosphate, glucose, and L-alanine) are consumed by the bacteria as apart of their normal metabolism. The fluorescent is moieties (i.e.,4-methylumberiferone or 7-amino-4-methyl coumarin) produce fluorescentsignals (maximum emission at 450 nm) which are used as indicators ofbacterial viability.

[0053] The time required for the fluorescent color to appear isdependent upon the concentration of bacteria present in the reagent.Higher concentration of viable bacteria in the medium results in aproportional decrease in the time required for color development.Therefore, this test can be adapted to instrumentation because of thelinear relationship between bacterial concentration and time to signaldevelopment, such as that described in Naqui et al., U.S. applicationSer. No. 08/201,110, hereby incorporated by reference.

[0054] Naqui et al. describes an accurate method for quantifying onenumber of bacteria in a liquid sample. The invention employs a novelapparatus for holding a liquid sample. The apparatus features a bagwhich is designed for receiving a liquid sample and subsequentlydistributes the liquid sample into separate compartments within the bagso that different aliquots of one or more sizes may be tested. Theinvention described in that application further allows quantifying themicroorganisms present in the sample by adding a medium to promotegrowth of microorganisms, heat sealing the bag of the invention forabout five seconds at a temperature of about 250° F. to 350° F.,incubating the sample at an appropriate temperature for an appropriatelength of time to allow growth of microorganisms, and recording andanalyzing the results. The quantifying step involves detecting thequantity and quality of the color change in each compartment, andcomparing that quantity and quality to a most probable number tablewhich relates that value to the bacterial concentration of the testsample.

[0055] For example, each 10 ml Quanti Tray™ system contains 50individual wells capable of holding 0.2 ml of medium. A 51st well ispresent which collects any “overfill” of medium not distributed into thefirst 50 wells. To begin the test the powder containing enzymesubstrates is first dissolved in 10 ml of sterile water. Next, thereagent is inoculated with a predetermined volume of homogenized foodmaterial. Finally, the reagent is sealed in a 10 ml Quanti Tray™ andplaced in a 35° C. incubator for 24 hours. The number of fluorescentwells present after incubation is compared against a most probablenumber (MPN) chart to determine the original concentration of bacteriapresent in the sample of food. Food containing higher than acceptableconcentrations of contaminating bacteria can be retested to verify theresults and/or disposed of to prevent distribution.

[0056] Because not all food is contaminated by the same bacteria foundin ground beef and chicken, other enzyme targets may need to be selectedto measure the total bacterial concentration of other types of food.

[0057] To design a medium for measuring the concentration of bacterialcontamination in a test sample from another type of food or othersources prone to bacterial contamination, methods known to those skilledin the art (including, but not limited to, plating, nucleic acidhybridization study, microscopic observation, etc.) are used to identifybacteria species existing in the sample. Once the bacteria species areidentified, one skilled in the art would be able to identify an enzymeor a group of enzymes that are characteristic of the bacteria species,and substrates acted on by the enzymes. Substrates having a nutrientmoiety and a detectable moiety linked together by a covalent bond thatis hydrolysed by the enzymes are produced to be used in the medium.

[0058] All publications referenced are incorporated by reference herein,including the nucleic acid sequences and amino acid sequences listed ineach publication. All the compounds disclosed and referred to in thepublications mentioned above are incorporated by reference herein,including those compounds disclosed and referred to in articles cited bythe publications mentioned above.

[0059] Other embodiments of this invention are disclosed in thefollowing claims.

What is claimed is:
 1. Method for detecting the existence or measuringthe concentration of bacteria in a test sample, comprising the steps of:providing a bacterial growth medium comprising three or more differentenzyme substrates, wherein each said substrate is hydrolysed by adifferent bacterial enzyme, and thereafter, causes or produces adetectable signal; inoculating said medium with said test sample andincubating said medium under a condition suitable for bacterial growthfor a certain time period; and detecting or measuring the detectablesignal as an indication of the existence or the concentration ofbacteria in said test sample.
 2. The method of claim 1, wherein saiddifferent substrates each having both a nutrient moiety and a detectablemoiety linked together by a covalent bond, and each said substrate ishydrolysed by a different bacterial enzyme to produce a separatedetectable moiety, and said separate detectable moiety causes orproduces a detectable signal.
 3. The method of claim 1, wherein saidbacteria are selected from the group consisting of Aeromonashydrophilia, Aeromonas caviae, Aeromonas sobria, Streptococcus uberis,Enterococcus faecium, Enterococcus faecalis, Bacillus sphaericus,Pseudomonas fluorescens, Pseudomonas putida, Serratia liquefaciens,Lactococcus lactis, Xanthomonas maltophilia, Staphylococcus simulans,Staphylococcus hominis, Streptococcus constellatus, Streptococcusanginosus, Escherichia coli, Staphylococcus aureus, Mycobacteriumfortuitum, and Klebsiella pneumonia.
 4. The method of claim 1, whereinsaid bacterial enzyme is selected from the group consisting of alkalinephosphatase, acid phosphatase, esterase, lipase,N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase,Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase,β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase,β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase,sulfatase, β-D-xylosidase, peptidase, (L or D aminoacid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin,and phosphohydrolase.
 5. The method of claim 1, wherein one of saidsubstrates is hydrolysed by a phosphatase enzyme, another of saidsubstrates is hydrolysed by a glycosidase enzyme, and a third saidsubstrate is hydrolysed by a peptidase enzyme.
 6. The method of claim 1,wherein said detectable moiety is a fluorescent moiety and saiddetectable signal is a fluorescent signal.
 7. The method of claim 1,wherein said substrates comprise 4-methylumbelliferyl phosphate,4-methylumbelliferyl-β-D-glucoside and L-alanine-7-amido-4-methylcoumarin.
 8. The method of claim 1, wherein said test sample is takenfrom a food product.
 9. The method of claim 8, wherein said food productis ground beef.
 10. The method of claim 8, wherein said food product ischicken.
 11. The method of claim 8, wherein said food product is water.12. The method of claim 1, wherein said medium is liquid.
 13. The methodof claim 1, wherein said time period is no more than 24 hours. 14.Method for detecting the existence or measuring the concentration ofbacteria in a test sample, comprising the steps of: providing abacterial growth medium comprising two or more different substrates,wherein each said substrate is hydrolysed by a different bacterialenzyme and thereafter causes or produces an identical type of detectablesignal; inoculating said medium with said test sample and incubatingsaid medium under a condition suitable for bacterial growth for acertain time period; and detecting or measuring the detectable signal asan indication of the existence or the concentration of bacteria in saidtest sample.
 15. The method of claim 14, wherein said differentsubstrates each having both a nutrient moiety and a detectable moietylinked together by a covalent bond, and each said substrate ishydrolysed by a different bacterial enzyme to produce a separatedetectable moiety, and said separate detectable moiety causes orproduces an identical type of detectable signal.
 16. The method of claim14, wherein said bacteria are selected from the group consisting ofAeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Streptococcusuberis, Enterococcus faecium, Enterococcus faecalis, Bacillussphaericus, Pseudomonas fluorescens, Pseudomonas putida, Serratialiquefaciens, Lactococcus lactis, Xanthomonas maltophilia,Staphylococcus simulans, Staphylococcus hominis, Streptococcusconstellatus, Streptococcus anginosus, Escherichia coli, Staphylococcusaureus, Mycobacterium fortuitum, and Klebsiella pneumonia.
 17. Themethod of claim 14, wherein said bacterial enzyme is selected from thegroup consisting of alkaline phosphatase, acid phosphatase, esterase,lipase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase,Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase,β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase,β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase,sulfatase, β-D-xylosidase, peptidase, (L or D aminoacid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin,and phosphohydrolase.
 18. The method of claim 14, wherein said enzyme isselected from the group consisting of a phosphatase enzyme, aglycosidase enzyme and a peptidase enzyme.
 19. The method of claim 14,wherein said detectable moiety is a fluorescent moiety and saiddetectable signal is a fluorescent signal.
 20. The method of claim 18 or19, wherein said substrates are selected from the group consisting of4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucoside andL-alanine-7-amido-4-methyl coumarin.
 21. The method of claim 14, whereinsaid test sample is taken from a food product.
 22. The method of claim21, wherein said food product is ground beef.
 23. The method of claim21, wherein said food product is chicken.
 24. The method of claim 21,wherein said food product is water.
 25. The method of claim 14, whereinsaid medium is liquid.
 26. The method of claim 14, wherein said timeperiod is no more than 24 hours.
 27. A bacterial growth mediumcomprising three or more different substrates, wherein each saidsubstrate is hydrolysed by a different bacterial enzyme, and thereafter,causes or produces a detectable signal.
 28. The medium of claim 27,wherein said different substrates each having both a nutrient moiety anda detectable moiety linked together by a covalent bond, and each saidsubstrate is hydrolysed by a different bacterial enzyme to produce aseparate detectable moiety, and said separate detectable moiety causesor produces a detectable signal.
 29. The medium of claim 27, whereinsaid bacterial enzyme is selected from the group consisting of alkalinephosphatase, acid phosphatase, esterase, lipase,N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase,Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase,β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase,β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase,sulfatase, β-D-xylosidase, peptidase, (L or D aminoacid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin,and phosphohydrolase.
 30. The medium of claim 27, wherein one of saidsubstrates is hydrolysed by a phosphatase enzyme, another of saidsubstrates is hydrolysed by a glycosidase enzyme, and a third saidsubstrate is hydrolysed by a peptidase enzyme.
 31. The medium of claim27, wherein said detectable moiety is a fluorescent moiety and saiddetectable signal is a fluorescent signal.
 32. The medium of claim 27,wherein said substrates comprise 4-methylumbelliferyl phosphate,4-methylumbelliferyl-β-D-glucoside and L-alanine-7-amido-4-methylcoumarin.
 33. The medium of claim 27, further comprising a test samplefrom a food product.
 34. The medium of claim 33, wherein said foodproduct is ground beef.
 35. The medium of claim 33, wherein said foodproduct is chicken.
 36. The medium of claim 33, wherein said foodproduct is water.
 37. The medium of claim 27, wherein said medium isliquid.
 38. A bacterial growth medium comprising two or more differentsubstrates, wherein each said substrate is hydrolysed by a differentbacterial enzyme and thereafter causes or produces an identical type ofdetectable signal.
 39. The medium of claim 38, wherein said differentsubstrates each having both a nutrient moiety and a detectable moietylinked together by a covalent bond, and each said substrate ishydrolysed by a different bacterial enzyme to produce a separatedetectable moiety, and said separate detectable moiety causes orproduces an identical type of detectable signal.
 40. The medium of claim38, wherein said bacterial enzyme is selected from the group consistingof alkaline phosphatase, acid phosphatase, esterase, lipase,N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase,Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase,β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase,β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase,sulfatase, β-D-xylosidase, peptidase, (L or D aminoacid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin,and phosphohydrolase.
 41. The medium of claim 38, wherein said enzyme isselected from the group consisting of a phosphatase enzyme, aglycosidase enzyme and a peptidase enzyme.
 42. The medium of claim 38,wherein said detectable moiety is a fluorescent moiety and saiddetectable signal is a fluorescent signal.
 43. The medium of claim 41 or42, wherein said substrates are selected from the group consisting of4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucoside andL-alanine-7-amido-4-methyl coumarin.
 44. The medium of claim 38, furthercomprising a test sample from a food product.
 45. The medium of claim44, wherein said food product is ground beef.
 46. The medium of claim44, wherein said food product is chicken.
 47. The medium of claim 44,wherein said food product is water.
 48. The medium of claim 38, whereinsaid medium is liquid.
 49. Method for detecting the existence ormeasuring the concentration of eukaryotic microbes in a test sample,comprising the steps of: providing a growth medium comprising three ormore different substrates, wherein each said substrate is hydrolysed bya different eukaryotic microbial enzyme and thereafter causes orproduces a detectable signal; inoculating said medium with said testsample and incubating said medium under a condition suitable formicrobial growth for a certain time period; and detecting or measuringthe detectable signal as an indication of the existence or theconcentration of eukaryotic microbes in said test sample.
 50. The methodof claim 49, wherein said different substrates each having both anutrient moiety and a detectable moiety linked together by a covalentbond, and each said substrate is hydrolysed by a different eukaryoticmicrobial enzyme to produce a separate detectable moiety, and saidseparate detectable moiety causes or produces a detectable signal. 51.The method of claim 49, wherein said eukaryotic microbes comprise ayeast.
 52. The method of claim 5 or 18, wherein said peptidase enzyme isan aminopeptidase enzyme.
 53. The medium of claim 30 or 41, wherein saidpeptidase enzyme is an aminopeptidase enzyme.